Method of identifying and treating invasive carcinomas

ABSTRACT

Prostasin protein has been found to be a useful marker for determination of the invasiveness of and as a means to treat human carcinomas. Using RT-PCR and western blot analyses, prostasin protein and mRNA expression were found in normal human prostate epithelial cells and the human prostate cancer cell line LNCaP, but not in the highly invasive human prostate cancer cell lines DU- 145 and PC-3. Imunohistochemistry studies of human prostate cancer specimens revealed a down-regulation of prostasin in high-grade tumors. Using RT-PCR and western blot analyses, prostasin protein and mRNA expression were found in a non-invasive human breast cancer cell line, MCF-7, while invasive human breast cancer cell lines MDA-MB-231 and MDA-MB-435s were found not to express either the prostasin protein or the mRNA. A non-invasive human breast cancer cell line, MDA-MB-453, was shown to express prostasin mRNA but not prostasin protein. Transfection of DU-145 and PC-3 cells with a full-length human prostasin cDNA restored prostasin expression and reduced the in vitro invasiveness by 68% and 42%, respectively. Transfection of MDA-MB-231 and MDA-MB-435s cells with a full-length human prostasin cDNA restored prostasin expression and reduced the in vitro invasiveness by 50% for either cell line.

[0001] This invention relates to prostasin and its use in the diagnosisand treatment of prostate and breast cancers, and claims the priority toU.S. Provisional Application Serial No. 60/174,801 filed Jan. 6, 2000and was supported in part by Department of Defense Prostate CancerResearch Grant DAMD17-98-1-8590, and in part by grants to principalinvestigator K. X. CHAI from the Florida Hospital Gala Endowed Programfor Oncologic Research.

BACKGROUND AND PRIOR ART

[0002] For men in the U.S., prostate cancer is the most commonlydiagnosed cancer, and the second leading cause of cancer-related death(Greenlee R. T., Murray T. Bolden S, Wingo Pa. Cancer statistics, 1999.Ca: a Cancer Journal for Clinicians 2000;50:7-33). Prostate cancersoriginate as localized lesions; some of these localized lesions willprogress to become invasive, migratory and metastatic. Our currentunderstanding of the mechanisms of the prostate cancer invasion process,however, is poor. Our ability to predict the acquisition of invasivepotentials by a prostate cancer is limited.

[0003] The mechanisms leading to the development of a prostate cancerare complex. Currently, it is believed to be the result of multipletransformation steps from normal prostate glandular cells (Carter H. B.Piantadosi S. Isaacs J. T. Clinical evidence for and implications of themultistep development of prostate cancer. Journal of Urology.143(4):742-6, 1990). The initial steps result in what are described asprostatic interepithelial neoplastic (PIN) lesions (Isaacs J. T.Molecular markers for prostate cancer metastasis. Developing diagnosticmethods for predicting the aggressiveness of prostate cancer. [Review][92 refs] American Journal of Pathology. 150(5):1511-21, 1997). ThesePIN lesions may then typically have three different fates based on anassessment of their impact to the patient. The PIN lesions can remain assuch, not producing histologically detectable prostate cancer, orfurther transform into histologically detectable prostate cancer. Mostof the histologically detectable prostate cancers will be asymptomaticin the patient and remain non-manifest clinically as many are discoveredpost-mortem (Carter H Coffey D. Prostate Cancer: the magnitude of theproblem in the United States. In a Multidisciplinary Analysis ofControversies in the Management of Prostate Cancer. (Eds. Coffey D.Resnick M. Door R. et al.), pp1-9, Plenum Press, 1988; Carter HBPiantadosi S. Issacs J. T. Clinical evidence for implications of themultistep development of prostate cancer. Journal of Urology.143(4):742-6,1990; Scardino PT. Weaver R. Hudson MA. Early detection ofprostate cancer. [Review] [102 refs] Human Pathology. 23(3):211-22,1999). Prostate cancers are diagnosed clinically by an estimate of sizeand location using the TNM staging system (Denis L J. Staging andprognosis of prostate cancer. European Urology. 24 Suppl 2:13-8, 1993),and by pathological staging based on an examination of the histology ofthe removed prostate via either biopsy or prostatectomy using a systemby D. F.Gleason, (Gleason DF. Classification of prostatic carcinomas.Cancer Chemotherapy Reports- Part 1. 50(3):125-8, 1966). About 50% ofprostate cancer cases receiving treatment are diagnosed clinically asadvanced, or, non-organ-confined (Scardino PT. Weaver R. Hudson MA.Early detection of prostate cancer. [Review] [102 refs] Human Pathology.23(3):211-22, 1992), for which no effective treatment exists (Yagoda A.Petrylak D. Cytotoxic chemotherapy for advanced hormone-resistantprostate cancer. [Review] [63 refs] Cancer. 71(3 Suppl): 1098-109, 1993and Petrylak, 1993). Of the remaining 50% cases, 1/3 (˜50,000) arediagnosed as organ-confined but micrometastasis may be present. Thefinal group of patients (˜100,000) have truly organ-confined prostatecancer and can be cured by radical prostatectomy (Sgrignoli AR Walsh PC.Steinberg GD. Steiner MS. Epstein JI. Prognostic factors in men withstage D 1 prostate cancer: identification of patients less likely tohave prolonged survival after radical prostatectomy [see comments].Journal of Urology. 152(4):1077-81, 1994 ; Zincke H. Oesterling JE.Blute ML. Bergstralh EJ. Myers RP. Barrett DM. long term (15 years)results after radical prostatectomy for clinically localized (stageT2cor lower) prostate cancer [see comments]. Journal of Urology. 152(5 Pt2): 1850-7, 1994) or left untreated (watchful waiting) without the riskof life-threatening or life-altering. For the patients withnon-organ-confined prostate cancers (discovered via either biopsy orsurgery), undergoing systemic treatment early is essential to themanagement of their cancer (Yagoda A. Petrylak D. Cytotixic chemotherapyfor advanced hormone-resistant prostate cancer [Review] [63 refs]Cancer. 71(3 Suppl): 1098-109, 1993). Consequently, it will be ideal,both medically and economically, if one could precisely predict uponearly pathological examination of the tumor, which group of patientswill have truly organ-confined disease versus which group will haveinvasive prostate cancer.

[0004] Clinical staging of prostate cancer generally depends on theresults of three tests that are performed in the following order: a PSA(prostate-specific antigen) blood test as a screening method; DRE(digital rectal examination) for an initial indication of palpabledisease; and, a biopsy to obtain samples for histological examination.Prostate cancers, removed either via biopsy or surgery, are gradedhistologically by the system of Gleason. (Gleason DF. Classification ofprostatic carcinomas. Cancer Chemotherapy Reports-Part1. 50(3):125-8,1966), which is an evaluation of how aggressive and howpoorly-differentiated the prostate cancers are. The aggressiveness ofprostate tumors: of low Gleason scores (<5) is limited; of high Gleasonscores (8-10) are highly aggressive; but, for the intermediateGleason-score (5-7) prostate cancers (76% of prostate tumors), theaccuracy of predicting their aggressiveness is poor (Gleason DF.Mellinger GT. Prediction of prognosis for prostatic adenocarcinoma bycombined histological grading and clinical staging. Journal of Urology.111 (1):58-64, 1974). Thus, the ability to accurately determine theaggressiveness of these intermediate Gleason-score prostate tumors hasremained as a practical challenge to, and a primary goal for, prostatecancer research (Isaacs JT. Molecular markers for prostate cancermetastasis. Developing diagnostic methods for predicting theaggressiveness of prostate cancer. [Review] [92 refs] American Journalof Pathology. 150(5):1511-21, 1997). Especially with regard to thenumber of patients (150,000) facing a decision of whether to undergosystemic treatment, the most urgent demand in prostate cancer care isthe development of methods to enhance our ability to accurately predictthe aggressiveness of the tumors with Gleason scores of 5-7.

[0005] It is now commonly believed that cancers occur via multipletransformation steps by accumulating mutations in three classes ofgenes: proto-oncogenes (Park M. Oncogenes. In The Genetic Basis of HumanCancer (Eds. Vogelstein B and Kinzler KW), pp205-28, McGraw-Hill HealthProfessions Divisions, 1998); tumor-suppressor genes (Knuutila S. AaltoY. Bjorkqvist AM. EL-Rifai W. Hemmer S. Huhta T. Kettunen E.Kiuru-Kuhlefelt S. Larramendy ML. Lushnikova T. Monni 0. Pere H. TapperJ. Tarkkanen M. Varis A. Wasenius VM. Wolf M. Zhu Y. DNA copy numberlosses in human neoplasms. [Review] [197 refs] American Journal ofPathology. 155(3):683-94, 1999); and, DNA repair genes (Knuutila S.Aslto Y. Bjorkqvit AM. EL-Rifai W. Hemmer S. Huhta T. Kettunen E.Kiuru-Kuhlefelt S. Larramedy ML. Lushnikova T. Monni 0. Pere H. TapperJ. Tarkkanen M. Varis A. Wasenius VM. Wolf M. Zhu Y. DNA copy numberlosses in human neoplasms. [Review] [197 refs] American Journal ofPathology. 155(3):683-94, 1999). The histological prostate cancers forwhich the prediction of clinical aggressiveness is difficult (those withthe intermediate Gleason scores 5-7) probably have not gone through thenecessary “multi-step” transformation to acquire the potentials tobehave aggressively (as would the high-grade cancers). This notion wassupported by studies comparing the course of prostate cancer developmentamong men in Japan and in the U.S., and Japanese men who migrated to theU.S. (Carter HB. Piantadosi S. Isaacs JT. Clinical evidence for andimplications of the multistep development of prostate cancer. Journal ofUrology, 1990; Haenszel W. Kurihara M. Studies of Japanese Migrants. I.Mortality form cancer and other diseases among Japanese in the UnitedStates. Journal of the National Cancer Institute.40(1):43-68, 1968;Akazaki K. Stemmerman GN. Comparartive study of latent carcinoma of theprostate among Japanese in Japan and Hawaii. Journal of the NationalCancer Institute. 50(5): 1137-44, 1973; Dunn JE. Cancer epidemiology inpopulations of the United States—with emphasis on Hawaii andCalifornia—and Japan. Cancer Research. 35(11 Pt.2 ):3240-5, 1975). Thefindings were that first- and second-generation Japanese men whomigrated to the U.S. have a higher prostate cancer incident rate thannative Japanese men. The emigrant Japanese men's prostate cancerincident rate is similar to that of men in the U.S. Investigating thechanges of expression in these three classes of cancer-relevant genesduring the course of prostate cancer development will lead to a betterunderstanding of the processes by which prostate cancers acquire theiraggressive potentials. The discovery of molecules whose changes can becorrelated to the staging of prostate cancer will provide new tools toimprove our ability to better predict the aggressive behaviors ofprostate cancer. This approach is now commonly referred to as “molecularstaging”.

[0006] Down-regulated genes such as tumor suppressors, invasionsuppressors or metastasis suppressors may be used as prostate cancermarkers. Examples of these genes that can potentially serve as prostatecancer markers for “molecular staging” are: KAI1 (Dong JT. Suzuki H. PinSS. Bova GS., Schalken JA, Issacs WB, Barrett JC. Issace IT.Down-regulation of the KAI1 metastasis suppressor gene during theprogression of human prostatic cancer infrequently involves genemutation or allelic loss. Cancer Research. 56(19): 4387-90, 1996); (UedaT, Ichikawa T., Tamaru j, Mikata A, Akakura K, Akimoto S, Imai T. Yoshie0. Shiraishi T. Yatani R. Ito H. Shimazaki J., Expression fof the KAI 1protein in benign prostatic hyperplasia and prostate cancer. AmericanJournal of Pathology 149(5): 1435-40, 1996); E-cadherin (Umbas R. IsaacsWB BringuierPP. Schaafsma HE. Karthaus HF. Oosterhof GO. Debruyne FM.Schalken JA. Decreased E-Cadherin expression is associated with poorprognosis is patients with prostate cancer. Cancer Research.52(18):5104-9, 1992, Umbas R. Isaacs WB. Bringuier PP. Schaafsma HE.Karthaus HF. Oosterhof GO Debruyne FM. Schalken JA. Decreased E-cadherinexpression is associated with poor prognosis in patients with prostatecancer. Cancer Research. 54(14):3929-33, 1994); β_(1C) integrin (FornaroM. Tallini G. Bofetiado CJ. Bosari S. Languino LR. Down-regulation of β1C integrin, an inhibitor of cell proliferation, in prostate carcinoma.American Journalof Pathology. 149(3):765-73,1996 Fornaro M. Manzotti M.Tallini G. Slear AE. Bosari S. Ruoslahti E. Languino LR. β1 C integrinin epithelial cells correlates with a nonproliferative phenotype: forcedexpression of β1 C inhibits prostate epithelial cell proliferation.American Journal of Pathology. 153(4):1079-87, 1998; p27(kip1) (TsihliasJ. Kapusta LR. DeBoer G. Morava-Protzner I. Zbieranowski I. BhattacharyaN. Catzavelos GC. Klotz LH. Slingerland JM. Loss of cyclin-dependentkinase inhibitor p27Kip1 is a novel prognostic factor in localized humanprostate adenocarcinoma. Cancer Research. 58(3):542-8, 1998); and CD44(Lou W. Krill D. Dhir R. Becich MJ. Dong JT. Frierson HF Jr. Isaacs WB.Isaacs JT. Gao AC. Methylation of the CD44 metastasis suppressor gene inhuman prostate cancer. Cancer Research. 59(10):2329-31, 1999). By usingthe method of immunohistochemistry, these genes were found to bedown-regulated in prostate cancer. KAI's down regulation is a potentialpredictor of metastasis (Dong, et al., 1996: Ueda et al., 1996). WhileEcadherin's down regulation is strongly correlated to higher Gleasongrades . Functional studies of these genes have given clues to theirrole in prostate cancer or normal prostate biology. (Debruyne FM. IsaacsWB. Expression of the cellular adhesion molecule Ecadherin is reduced orabsent in high-grade prostate cancer. Cancer Research. 52(18):5104-9,1992; Umbas R. Isaacs WB. Bringuier PP. Schaafsma HE. Karthaus HF.Oosterhof GO Debruyne FM. Schalken JA. Decreased E-cadherin expressionis associated with poor prognosis in patients with prostate cancer.Cancer Research. 54(14):3929-33, 1994; metastasis suppressor gene duringthe progression of human prostatic cancer infrequently involves genemutation or allelic loss. Cancer Research. 56(19):4387-90; 1996 Ueda T.Ichikawa T. Tamaru J. Mikata A. Akakura K. Akimoto S. Imai T. Yoshie 0.Shiraishi T. Yatani R. Ito H. Shimazaki J. Expression of the KAI1protein in benign prostatic hyperplasia and prostate cancer. AmericanJournal of Pathology. 149(5):1435-40, 1996) while E-cadherin'sdown-regulation is strongly correlated to higher Gleason grades (UmbasR. Schalken JA. Aalders TW. Carter BS. Karthaus HF. Schaafsma HE.Debruyne FM. Isaacs WB. Expression of the cellular adhesion moleculeE-cadherin is reduced or absent in high-grade prostate cancer. CancerResearch. 52(18):5104-9,1992; Umbas R. Isaacs WB. Bringuier PP.Schaafsma HE. Karthaus HF. Oosterhof GO. Debruyne FM. Schalken JA.Decreased E-cadherin expression is associated with poor prognosis inpatients with prostate cancer. Cancer Research. 54(14):3929-33, 1994)Functional studies of these genes have given clues to their role inprostate cancer or normal prostate biology. Human KAI suppressmetastasis of rat prostate cancer cells upon gene transfer (Dong JT.Lamb PW. Rinker-Schaeffer CW. Vukanovic J. Ichikawa T. Isaacs JT.Barrett JC. KAI1, a metastasis suppressor gene for prostate cancer onhuman chromosome 11p11.2 [see comments]. Science. 268(5212):884-6,1995). β_(1C) and p27(kip₁) are involved in signaling pathway thatinhibits cell proliferation (Fomaro M. Tallini G. Zheng DQ. Flanagan WM.Manzotti M. Languino LR. p27(kip1) acts as a downstream effector of andis coexpressed with the betal C integrin in prostatic adenocarcinoma.Journal of Clinical Investigation. 103(3):321-9, 1999). E-cadherinexpression is progressively lost during the transformation of ratprostate cancer from non-invasive to invasive (Bussemakers MJ. vanMoorselaar RJ. Giroldi LA. Ichikawa T. Isaacs JT. Takeichi M. DebruyneFM. Schalken JA. Decreased expression of E-cadherin in the progressionof rat prostatic cancer. Cancer Research. 52(10):2916-22, 1992), and ithas also been shown to be an invasion suppressor (Lou W. Krill D. DhirR. Becich MJ. Dong JT. Frierson HF Jr. Isaacs WB. Isaacs JT. Gao AC.Methylation of the CD44 metastasis suppressor gene in human prostatecancer. Cancer Research. 59(10):2329-31, 1999). CD44 loss of expressionis associated with high metastatic ability and transfection of CD44suppresses metastasis without affecting tumorigenecity of rat prostatecancer cells (Gao AC. Lou W. Dong JT. Isaacs JT. CD44 is a metastasissuppressor gene for prostatic cancer located on human chromosome 11p13.Cancer Research. 57(5):846-9, 1997). CD44 down-regulation is aprognostic marker for prostate cancer (Noordzij MA. van Steenbrugge GJ.Verkaik NS. Schroder FH. van der Kwast TH. The prognostic value of CD44isoforms in prostate cancer patients treated by radical prostatectomy.Clinical Cancer Research. 3(5):805-15, 1997).

[0007] Human prostate cancer histology is heterogeneous, or“multi-focal” in nature (Isaacs JT. Bova GS. Prostate Cancer. In TheGenetic Basis of Human Cancer (Eds. Vogelstein B and Kinzler KW),pp653-60, McGraw-Hill Health Professions Division, 1998 and Bova, 1998),with an average of five lesions in a patient (Bastacky SI. Wojno KJ.Walsh PC. Carmichael MJ. Epstein JI. Pathological features of hereditaryprostate cancer. Journal of Urology. 153(3 Pt 2):987-92, 1995). Betweenthe tumor regions of a prostate and within a single tumor region, thegenetic causes to the cancer are heterogeneous and independent as well(Sakr WA. Macoska JA. Benson P. Grignon DJ. Wolman SR. Pontes JE.Crissman JD. Allelic loss in locally metastatic, multisampled prostatecancer. Cancer Research. 54(12):3273-7, 1994; Qian J. Bostwick DG.Takahashi S. Borell TJ. Herath JF. Lieber MM. Jenkins RB. Chromosomalanomalies in prostatic intraepithelial neoplasia and carcinoma detectedby fluorescence in situ hybridization. Cancer Research. 55(22):5408-14,1995; Mirchandani D. Zheng J. Miller GJ. Ghosh AK. Shibata DK. Cote RJ.Roy-Burman P. Heterogeneity in intratumor distribution of p53 mutationsin human prostate cancer. American Journal of Pathology. 147(1):92-101,1995). In the end, the best predictor of prostate cancer's potential togain invasiveness may be a consideration of a number of genes whoseexpression levels change along the course of cancer development, asdemonstrated in principle by Greene et al. (Greene GF. Kitadai Y.Pettaway CA. von Eschenbach AC. Buucana CD. Fidler IJ. Correlation ofmetastasis-related gene expression with metastatic potential in humanprostate carcinoma cells implanted in nude mice using an in situmessenger RNA hybridization technique. American Journal of Pathology.150(5):1571-82, 1997). Hence, expanding the repertory of such genes,including both the onco-genes and the suppressor genes, will enhance theaccuracy and dependability of this approach.

[0008] As for the treatment options of prostate cancer, patients withtruly organ-confined prostate cancer can be cured by radicalprostatectomy. Patients with non-organ-confined prostate cancers,however, have very low survival rates and the current treatments arelargely ineffective (Yagoda A. Petrylak D. Cytotoxic chemotherapy foradvanced hormone-resistant prostate cancer. [Review] [63 refs] Cancer.71(3 Suppl): 1098-109, 1993).

[0009] Breast cancer is the most diagnosed cancer in women and thesecond leading cancer related cause of death in women (Greenlee RT,Murray T, Bolden S, Wingo PA. Cancer statistics, 1999. Ca: a CancerJournal for Clinicians 2000;50:7-33). Breast ductal carcinoma in situ(hereinafter indicated as DCIS) incidence has increased dramaticallysince 1983 as a result of implementing screening programs (Ernster VL.Barclay J. Increases in ductal carcinoma in situ (DCIS) of the breast inrelation to mammography: a dilemma. [Review] [34 refs] Journal of theNational Cancer Institute. Monographs. (22): 151-6, 1997). DCIS isdescribed as a malignant growth of epithelial cells within the ducts andlobules of the breast, and is believed to be the precursor of allinvasive breast carcinoma. DCIS itself is non-life-threatening; however,current treatment options for DCIS include masectomy, lumpectomy,radiotherapy or tamoxifen (Emster VL. Barclay J. Increases in ductalcarcinoma in situ (DCIS) of the breast in relation to mammography: adilemma. [Review] [34 refs] Journal of the National Cancer Institute.Monographs. (22): 151-6, 1997; Hwang ES. Esserman LJ. Management ofductal carcinoma in situ. [Review] [91 refs] Surgical Clinics of NorthAmerica. 79(5):1007-30, viii, 1999). These treatment options for DCISare at best controversial due primarily to a lack of precision indiagnosis and prognosis of whether the detected DCIS will progress toinvasive breast cancer and whether recurrence is likely after treatment,usually with a high percentage being invasive breast cancer (Zaugg K.Bodis S. Is there a role for molecular prognostic factors in theclinical management of ductal carcinoma in situ (DCIS) of the breast?.[Review] [49 refs] Radiotherapy & Oncology. 55(2):95-9, 2000). Atpresent, histological grading (nuclear grading and whether come do-typenecrosis is present) and the size of the DCIS are used to provideassessments of risk of DCIS to progress into invasive breast cancer(Zaugg K. Bodis S. Is there a role for molecular prognostic factors inthe clinical management of ductal carcinoma in situ (DCIS) of thebreast?. [Review] [49 refs] Radiotherapy & Oncology. 55(2):95-9, 2000;Shoker BS. Sloane JP. DCIS grading schemes and clinical implications.[Review] [40 refs] Histopathology. 35(5):393-400, 19; van de Vijver MJ.Ductal carcinoma in situ of the breast: histological classification andgenetic alterations. [Review] [69 refs] Recent Results in CancerResearch. 152:12334, 1998). These parameters are far from being adequatefor making the most accurate choice of treatment, resulting in a choiceeither overly excessive or conservative, in either case, the patientwill suffer unnecessarily. Molecular markers can help improve ourability to better diagnose DCIS and stratify treatment options,especially the molecular markers that, themselves, play a role in theprogression of DCIS to invasive breast cancer (Zaugg K. Bodis S. Isthere a role for molecular prognostic factors in the clinical managementof ductal carcinoma in situ (DCIS) of the breast?. [Review] [49 refs]Radiotherapy & Oncology. 55(2):95-9, 2000; Silverstein MJ. Masetti R.Hypothesis and practice: are there several types of treatment for ductalcarcinoma in situ of the breast?. [Review] [53 refs] Recent Results inCancer Research. 152:105-22, 1998). The tumorigenesis process is amulti-step transformation in which molecular events escalate to thefinal stage of invasive phenotype (Silverstein MJ. Masetti R. Hypothesisand practice: are there several types of treatment for ductal carcinomain situ of the breast?. [Review] [53 refs] Recent Results in CancerResearch. 152:105-22, 1998). The conventional paradigm of proteaseinvolvement in the development and progression of cancer has been theassignment of a usually negative role to the proteases, such aspromoting tumor invasion (Mignatti P. Rifkin DB. Biology andbiochemistry of proteinases in tumor invasion. [Review] [306 refs]Physiological Reviews. 73(1):161-95, 1993). In turn, the conventionalparadigm of protease inhibitors in relation to cancer is usually regardof a beneficial effect for the presence of these molecules (Kennedy AR.Chemopreventive agents: protease inhibitors. Pharmacol Therapeut78:167-209, 1998). Recently, however, the picture of a new paradigm isbeginning to emerge for several serine proteases in breast, prostate,and testicular cancers. A “normal epithelial cell specific-1” (NESI)serine protease was found to be down-regulated in breast and prostatecancers, and it functions as a tumor suppressor (Goyal J, Smith KM,Cowan JM, Wazer DE, Lee SW, Band V. The role for NES 1 serine proteaseas a novel tumor suppressor. Cancer Res 58:4782-4786, 1998). Aprostate-specific serine protease, prostase (Nelson PS. Gan L. FergusonC. Moss P. Gelinas R. Hood L. Wang K. Molecular cloning andcharacterization of pro stase, an androgen-regulated serine proteasewith prostaterestricted expression. Proceedings of the National Academyof Sciences of the United States of America. 96(6):3114-9, 1999), wasshown to be expressed in normal prostate but not in prostate cancer celllines DU-145 and PC-3. The expression of a testis-specific serineprotease, testisin, was shown to be lost in testicular cancer througheither a loss of gene (Hooper JD. Nicol DL. Dickinson JL. Eyre HJ.Scarman AL. Normyle JF. Stuttgen MA. Douglas ML. Loveland KA. SutherlandGR. Antalis TM. Testisin, a new human serine proteinase expressed bypremeiotic testicular germ cells and lost in testicular germ celltumors. Cancer Research. 59(13):3199-205, 1999) or methylation in thepromoter (Boucaut K, Douglas M, Clements J, Antalis T. The serineproteinase testisin may act as a tumor and/or growth suppressor in thetestis and may be regulated by DNA methylation. Cancer Genetics andTumor Suppressor Genes, Cold Spring Harbor Laboratory, 2000). Further,transfection of human testicular cancer cells with a testisin cDNAreduced the tumor growth of xenografts of these cells in nude mice,suggesting a tumor suppressor function for testisin (Boucaut K, DouglasM, Clements J, Antalis T. The serine proteinase testisin may act as atumor and/or growth suppressor in the testis and may be regulated by DNAmethylation. Cancer Genetics and Tumor Suppressor Genes, Cold SpringHarbor Laboratory, 2000). The testisin serine protease is potentiallymembrane-bound as suggested by its structure and confirmed byimmunohistochemistry gene (Hooper JD. Nicol DL. Dickinson JL. Eyre HJ.Scarman AL. Normyle JF. Stuttgen MA. Douglas ML. Loveland KA. SutherlandGR. Antalis TM. Testisin, a new human serine proteinase expressed bypremeiotic testicular germ cells and lost in testicular germ celltumors. Cancer Research. 59(13):3199-205, 1999.) Prostasin serineprotease is an acidic protein (pI 4.5-4.8) of approximately 40 kDa inmolecular mass (Yu JX. Chao L. Chao J. Prostasin is a novel human serineproteinase from seminal fluid. Purification, tissue distribution, andlocalization in prostate gland. Journal of Biological Chemistry.269(29):18843-8, 1994). It is predominantly made in the prostate gland(˜140 ng/mg protein), with lesser amounts (2-6 ng/mg protein) also foundin the bronchi, colon, kidney, liver, lung, pancreas, and the salivaryglands (Yu JX. Chao L. Chao J. Prostasin is a novel human serineproteinase from seminal fluid. Purification, tissue distribution, andlocalization in prostate gland. Journal of Biological Chemistry.269(29):18843-8, 1994). Prostasin is secreted in the prostatic fluid,and can be detected in the semen (˜9 μg/ml). Prostasin expression islocalized to the epithelial cells of human prostate gland by in situhybridization histochemistry using an antibody or an anti-sense RNAprobe (Yu JX. Chao L. Chao J. Prostasin is a novel human serineproteinase from seminal fluid. Purification, tissue distribution, andlocalization in prostate gland. Journal of Biological Chemistry.269(29): 18843-8, 1994; (Yu JX. Chao L. Chao J. Molecular cloning,tissue-specific expression, and cellular localization of human prostasinmRNA. Journal of Biological Chemistry. 270(22): 13483-9, 1995).Molecular cloning of a full-length human prostasin cDNA revealed thatits predicted amino acid residue sequence contains a carboxyl-terminalhydrophobic region that can potentially anchor the protein on themembrane (Yu JXChao L. Chao J. Molecular cloning, tissue-specificexpression, and cellular localization of human prostasin mRNA. Journalof Biological Chemistry. 270(22):13483-9, 1995). At the amino acidlevel, prostasin is similar to plasma kallikrein, coagulation factor XI,hepsin, plasminogen, acrosin, prostase, and, in particular, testisin(sharing 44% sequence identity) [Nelson PS. Gan L. Ferguson C. Moss P.Gelinas R. Hood L. Wang K. Molecular cloning and characterization ofprostase, an androgen-regulated serine protease with prostate-restrictedexpression. Proceedings of the National Academy of Sciences of theUnited States of America. 96(6):3114-9, 1999; Hooper JD. Nicol DL.Dickinson JL. Eyre HJ. Scarman AL. Normyle JF. Stuttgen MA. Douglas ML.Loveland KA. Sutherland GR. Antalis TM. Testisin, a new human serineproteinase expressed by premeiotic testicular germ cells and lost intesticular germ cell tumors. Cancer Research. 59(13):3199-205, 1999; YuJX. Chao L. Chao J. Molecular cloning, tissue-specific expression, andcellular localization of human prostasin mRNA. Journal of BiologicalChemistry. 270(22):13483-9, 1995). A membrane-bound Xenopus kidneyepithelial cell sodium channel-activating protease (CAP 1) was shown tobe highly homologous to human prostasin as well (sharing 53% sequenceidentity at the amino acid level) (Vallet V. Chraibi A. Gaeggeler HP.Horisberger JD. Rossier BC. An epithelial serine protease activates theamiloride-sensitive sodium channel. Nature. 389(6651):607-10, 1997).Prostasin is encoded by a single-copied gene, which is located on humanchromosome 16p11.2 (Yu JX. Chao L. Ward DC. Chao J. Structure andchromosomal localization of the human prostasin (PRSS8) gene. Genomics.32(3):334-40, 1996). The secreted prostasin cleaves synthetic substratesin vitro preferentially at the carboxyl-terminal side of Arg residue,and is thus considered a trypsin-like serine protease (Yu JX. Chao L.Chao J. Prostasin is a novel human serine proteinase from seminal fluid.Purification, tissue distribution, and localization in prostate gland.Journal of Biological Chemistry. 269(29):18843-8, 1994). Thephysiological function of prostasin, however, has remained unknown. Bycomparing gene expression of normal tissues, pre-invasive cancer, andinvasive cancer, it would be highly advantageous to discover molecularmarkers that display a differential expression pattern between thepre-invasive and the invasive phenotypes and use these markers for amore precise diagnosis and prognosis of prostate and/or breast cancers.With the enhanced precision in diagnosis and prognosis, treatmentoptions for patients with DCIS could then be stratified. Those with lowrisks of developing invasive breast cancer will have a higher confidencein choosing breast-conserving options, while those at high risks willponder more aggressive options with the necessary follow-up treatments.Similarly, those males with low risks of developing invasive prostatecancer will have a higher confidence in choosing prostate-conservingoptions, while those at high risks will ponder more aggressive optionswith the necessary follow-up treatments

SUMMARY OF THE INVENTION

[0010] The first objective of the present invention is to reducedeficiencies in the prior art with specific regard to differentialdiagnosis of invasive prostate and breast cancers and to treatment ofinvasive and metastatic prostate and breast cancers.

[0011] The second objective of the present invention is to provide a newmarker for prostate and breast cancer.

[0012] The third objective of the invention is to provide as a drug topatients with carcinoma of the prostate via delivery of a functionalprostasin gene.

[0013] The fourth objective of the invention is to provide as a drug topatents with carcinoma of the prostate via delivery of a functionalprostasin cDNA.

[0014] The fifth objective of the invention is to provide as a drug topatents with carcinoma of the breast via delivery of a functionalprostasin gene.

[0015] The sixth objective of the invention is to provide as a drug topatents with carcinoma of the breast via delivery of a functionalprostasin cDNA.

[0016] This invention identifies prostasin serine protease as apotential marker, and as a tumor invasion suppressor for prostate andbreast cancers and thus provides methods (a), (b), and (c) ofdetermining invasiveness levels of human carcinomas:

[0017] (a) using prostasin protein levels, comprising the steps of:sampling a human carcinoma tissue; determining prostasin protein levelsin the human carcinoma tissue; preferably by applying an immunologicalreagent-antibody to said tissue wherein the reagent-antibody becomesbound to prostasin protein in said tissue, and determining invasivenessof the human carcinoma tissue based on the prostasin protein levels:. or

[0018] (b) using prostasin mRNA levels, comprising the steps of:sampling a human carcinoma tissue; determining prostasin mRNA levels inthe human carcinoma tissue preferably by applying prostasin-specificanti-sense RNA probes in an in situ hybridization to determine theprostasin mRNA levels in the separated human carcinoma tissue thedetermination of the prostasin mRNA levels in the separated humancarcinoma sample tissue; to make possible and determining invasivenessof the human carcinoma tissue based on the prostasin mRNA levels; or

[0019] (c) (c) using prostasin gene promoter DNA methylation levels,comprising the steps of: sampling a human carcinoma tissue; determiningprostasin gene promoter DNA methylation levels in the human carcinomatissue, preferably by applying prostasin-promoterspecificoligonucleotide primers in a PCR to determine the prostasin genepromoter DNA methylation levels in the sampled human carcinoma tissueand determining invasiveness of the human carcinoma tissue based on theprostasin gene promoter DNA methylation levels,

[0020] as well as a method of treating invasive human carcinomascomprising the steps of: incorporating human prostasin nucleic acid intoa selected gene delivery vector nucleic acid to form a recombinantnucleic acid; preferably wherein the nucleic acid includes a gene orcDNA delivering the recombinant nucleic acid into a human carcinoma; andreducing invasiveness of the human carcinoma.

[0021] Further objects and advantages of this invention will be apparentfrom the following detailed description of a presently preferredembodiment, which is illustrated schematically in the accompanyingdrawings.

BRIEF DESCRIPTION OF THE FIGURE

[0022]FIG. 1 is a diagrammatic representation of the analysis ofprostasin indicative of the absence or existence, or of the invasivenessof human prostate carcinoma.

[0023]FIG. 2 is a diagrammatic representation of the analysis ofprostasin indicative of the absence or existence, or of the invasivenessof human mammary carcinoma.

[0024]FIG. 3 shows human prostasin expression in prostate epithelialcells.

[0025]FIG. 4A shows immunohistochemical detection of prostasin proteinin benign human prostate tissues.

[0026]FIG. 4B is an enlarged view of the boxed region of FIG. 4A.

[0027]FIG. 4C shows immunohistochemistry of benign human prostatetissues with prostasin antibody omitted in the procedures, no epithelialcells displayed any staining.

[0028]FIG. 4D is an enlarged view of the boxed region of FIG. 4C.

[0029]FIG. 4E shows immunohistochemistry of human prostate tumor withprostasin antibody omitted in the procedures, no epithelial cellsdisplayed any staining.

[0030]FIG. 4F shows immunohistochemical detection of prostasin proteinin benign human prostate tissue surrounded by prostate carcinomas.

[0031]FIG. 4G shows immunohistochemical detection of prostasin proteinin Gleason grade 2 prostate tumor.

[0032]FIG. 4H is an enlarged view of the boxed region of FIG. 4G.

[0033]FIG. 4I shows immunohistochemical detection of prostasin proteinin Gleason grade 3 prostate tumor.

[0034]FIG. 4J is an enlarged view of the boxed region of FIG. 4I.

[0035]FIG. 4K shows immunohistochemical detection of prostasin proteinin Gleason grade 4 prostate tumor.

[0036]FIG. 4L is an enlarged view of the boxed region of FIG. 4K.

[0037]FIG. 5 shows human prostasin expression in human breast cancercell lines.

[0038]FIG. 6 shows promoter hypermethylation of the human prostasin genein human prostate and breast cancer cell lines.

[0039]FIG. 7 shows prostasin protein expression and in vitro invasiveproperties of the DU-145 and the PC-3 transfectants.

[0040]FIG. 8 shows prostasin protein expression and in vitro invasiveproperties of the MDA-MB-231 and the MDA-MB-435s transfectants.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0041] Before explaining the disclosed embodiment of the presentinvention in detail it is to be understood that the invention is notlimited in its application to the details of the particular arrangementshown since the invention is capable of other embodiments. Also, theterminology used herein is for the purpose of description and not oflimitation.

[0042] Using RT-PCR and western blot analyses, prostasin protein andmRNA expression were found in normal human prostate epithelial cells andthe human prostate cancer cell line LNCaP, but discovered not present inthe highly invasive human prostate cancer cell lines DU-145 and PC-3.Immunohistochemistry studies of human prostate cancer specimens revealeda down-regulation of prostasin in high-grade tumors.

[0043] Using RT-PCR and western blot analyses, prostasin protein andmRNA expression were found in a non-invasive human breast cancer cellline, MCF-7, while invasive human breast cancer cell lines MDA-MB-231and MDA-MB-435s were also discovered not to express either the prostasinprotein or the mRNA. A non-invasive human breast cancer cell line,MDA-MB-453, was shown to express prostasin mRNA but not prostasinprotein. Examination of the prostasin gene promoter in the humanprostate and breast cancer cell lines by Southern blot analysis revealedheterogeneous methylation of the promoter in DU-145, PC-3 and MDA-MB-453cells, and homogeneous methylation of the promoter in MDA-MB-231 andMDA-MB-435s cells. The prostasin gene promoter in normal human prostateepithelial cells, the LNCaP and the prostasin gene promoter in the humanprostate and breast cancer cell lines by Southern blot analysis revealedheterogeneous methylation of the promoter in DU-145, PC-3 and MDA-MB-453cells, and homogeneous methylation of the promoter in MDA-MB-231 andMDA-MB-435s cells. The prostasin gene promoter in normal human prostateepithelial cells, the LNCaP and the MCF-7 cells was shown to beunmethylated. Transfection of DU-145 and PC-3 cells with a full-lengthhuman prostasin cDNA restored prostasin expression and reduced the invitro invasiveness by 68% and 42%, respectively. Transfection ofMDA-MB-231 and MDA-MB-435s cells with a full-length human prostasin cDNArestored prostasin expression and reduced the in vitro invasiveness by50% for either cell line. Cell proliferation was unaffected byre-expression of prostasin. Our data indicate that prostasin isimplicated in normal prostate biology and its down-regulation inprostate cancer, and its absence in invasive prostate and breast cancercell lines indicates increased invasiveness. Our results also indicatethat delivering a functional human prostasin gene to invasive prostateand breast cancers can reduce the invasiveness.

[0044] For a facile understanding of the invention embodied herein,reference should now be made to FIGS. 1 and 2. FIG. 1 sets forth aschematic for determining if a male has prostate cancer. Human blood 10is taken from the male and analyzed for the presence of prostasin serineprotease 14. If NO 12 prostasin is found, there is probably little or nocancer. If there is a presence YES 16 of prostasin serine protease 14,there is prostate cancer.

[0045] If a biopsy of the prostate gland 20 is analyzed for the presenceof a normal amount YES 22 of prostasin serine protease 14, there is nocancer or there is non-invasive cancer. If there is NO 24 or Reduced 26amount of prostasin serine protease 14, there is invasive cancer.

[0046]FIG. 2 sets forth a schematic for determining if a female hasbreast cancer. Human blood 100 is taken from the female and analyzed forthe presence of prostasin serine protease 140. If NO 120 prostasin isfound, there is prospectively no cancer. If there is a presence YES 160of prostasin serine protease 140, there is breast cancer. If a biopsy ofthe breast 200 is analyzed for the presence of YES 220 of prostasinserine protease 140, there is no invasive cancer but may be non-invasivecancer. If there is NO 240 or Reduced 260 amount of prostasin serineprotease 140, there is invasive cancer.

[0047] The levels of prostasin protein in the epithelial cells of thehuman prostate gland can be used as a diagnostic marker for thepotential invasiveness of prostate tumors. The supporting evidence camefrom our findings that two invasive human prostate cancer cell linesDU-145 and PC-3 do not express prostasin while normal prostateepithelial cells and a non-invasive prostate cancer cell line LNCaPexpress both the prostasin mRNA and protein

[0048] Refer now to FIG. 3 which shows human prostasin expression inprostate epithelial cells. By means of western blot analysis (upperpanel), prostasin (as a 40-kDa band) was detected in normal humanprostate epithelial cells (CC-2555) and the LNCaP cells, but not in theDU-145 or PC-3 cells. An equal amount of total protein (100 fig) wasloaded for each sample. At the mRNA level, human prostasin mRNA (via a232-bp amplified DNA band) was detected in normal prostate epithelialcells (CC-2555) and the LNCaP cells, but not in the DU145 or PC-3 cellsas analyzed by RT-PCR/Southern blot hybridization (middle panel).Co-amplification of a 556-bp human P-actin message (as shown in the gelphotograph in the lower panel) confirmed the quality and the quantity ofthe RNA applied in each RT-PCR.

[0049] Expression of prostasin protein is reduced in high-grade humanprostate tumor. Prostatectomy specimens from 39 patients (128 sections)were subjected to immunohistochemistry using a prostasin-specificantibody. Overall, in non-tumor or benign prostate epithelia, 89.0% ofthe examined areas demonstrated positive staining for prostasin proteinand 11.0% were considered negative (based on the scoring system used forHercepTest™, DAKO Corporation, Carpinteria, Calif.). In all tumorspecimens that were examined, prostasin was detected in 93.3% of the lowGleason grade areas (<grade 2), 44.4% of Gleason grade 3 areas, 21.1% ofGleason grade 4 areas, but not in Gleason grade 5 areas (data summarizedin Table 1). The mean prostasin immunostaining score was foundsignificantly decreased in high-grade prostate tumors as compared tonontumor areas (ANOVA, p<0.0001).

[0050] Representative staining images of non-tumor (benign) areas andprostate tumor areas are shown in FIG. 4a-4 l, which providesimmunohistochemical detection of prostasin protein in tissues.Paraffin-embedded human prostate sections were stained for prostasinprotein expression evaluation using a specific antibody as described (YuJX. Chao L. Chao J. Prostasin is a novel human serine proteinase fromseminal fluid. Purification, tissue distribution, and localization inprostate gland. Journal of Biological Chemistry. 269(29):18843-8, 1994).Prostasin positive staining (brown color) was detected in the cytoplasmand apical membrane in non-tumor or benign epithelial cells.

[0051] The prostasin protein was detected in the cytoplasm and on theplasma membrane (apical) of benign epithelial cells lining the secretorylumen as well as in the secretion inside the lumen (FIG. 4A and 4B,score 3, or +++), confirming the results of (Yu JX. Chao L. Chao J.Prostasin is a novel human serine proteinase from seminal fluid.Purification, tissue distribution, and localization in prostate gland.Journal of Biological Chemistry. 269(29):18843-8, 1994). When apre-immune rabbit serum was used in place of the prostasin antiserum, nostaining was observed in either the non-tumor epithelia (FIG. 4C and 4D)or tumor epithelia (FIG. 4E). Tumor epithelia displayed various degreesof prostasin immunostaining as shown in FIG. 4F-4L. In Gleason grade 1-2tumors, moderate prostasin staining is seen in the cytoplasm and on theplasma membrane of some epithelial cells, as well as in the secretion inthe lumen (FIG. 4G and 4H, score 2, or ++). In Gleason grade 3 tumors, alesser number of epithelial cells displayed the moderate level prostasinstaining (FIG. 41 and 4J). In Gleason grade 4 tumors, most epithelialcells did not show any prostasin staining, while some prostasin stainingcan be seen in rare, sporadic tumor cells (FIG. 4K and 4L, as indicatedby the arrow, score 0). Genetically, prostate tumors are heterogeneousand multi-focal in nature, in that one patient's gross-anatomy tumorcomes from multiple initial lesions which are caused by differentinitial transformation events and progress to different stages bydifferent ensuing transformations (Isaacs JT. Bova GS. Prostate Cancer.In The Genetic Basis of Human Cancer (Eds. Vogelstein B and Kinzler KW),pp653-60, McGraw-Hill Health Professions Division, 1998). The Gleasongrading, when used as a percentage of each cancer occupied by Gleasongrade 4/5 areas, is independently associated with prostate cancerprogression (Stamey TA, McNeal JE, Yemoto CM, Sigal BM, Johnstone IM.Biological determinants of cancer progression in men with prostatecancer. JAMA 281:1395-1400, 1999). We found a significant decrease ofprostasin expression in the high-grade, i.e., the more progressivelytransformed tumors.

[0052] As earlier indicated, FIG. 5 shows human prostasin expression inhuman breast cancer cell lines. By means of western blot analysis (upperpanel), prostasin (as a 40-kDa band) was detected in MCF-7 cells, butnot in MDA-MB-453, MDA-MB-231, or MDAMB-435s cells. An equal amount oftotal protein (100 μg) was loaded for each sample. At the mRNA level,human prostasin mRNA (via a 232-bp amplified DNA band) was detected inMCF-7 and MDA-MB-453 cells, but not in the MDA-MB-231 or MDA-MB435scells as analyzed by RT-PCR/Southern blot hybridization (lower panel).

[0053] Prostasin mRNA expression is seen absent in two invasive humanbreast cancer cell lines while two non-invasive breast cancer cell linesexpress prostasin mRNA or protein. Analysis of prostasin expression inhuman breast cancer cell lines showed that the non-invasive MCF-7 andMDA-MB-453 cells express the prostasin mRNA while the highly invasiveMDA-MB-231 and MDA-MB-435s cells do not express the prostasin mRNA (seeFIG. 5). Expression of prostasin mRNA in normal human breast can bedemonstrated by the presence of two GenBank normal human breast ESTsequences coding for prostasin (Accession numbers R48653, and R48557).The MCF-7 cells also express the prostasin protein as determined bywestern blot analysis (again see FIG. 5).

[0054] Prostasin down-regulation in prostate or breast cancer can becaused by promoter methylation or gene-specific mutation. Prostasinexpression decreases with increasing prostate cancer grade and is absentin invasive prostate and breast cancer cell lines. The chromosomal locuswhere the human prostasin gene is, 16p11.2, however, is not known to bean LOH hot-spot in prostate cancer or in breast cancer. Epigeneticevents (such as DNA methylation) may be an alternative mechanism of lossof expression for tumor suppressors or invasion suppressors. Refer nowto FIG. 6 which shows promoter hypermethylation of the human prostasingene in human prostate and breast cancer cell lines. Genomic DNA (5 μg)from the various cell lines (as indicated in the figure) were digestedwith the following restriction enzyme combinations, Xho I/BamH I(X/B,flanking cuts of the methylation-sensitive site), Xho I/BamH I/Msp I(X/B/M), or Xho I/BamH I/Hpa II (X/B/H). The digests were resolved in a0.8% agarose gel and transferred to an Immobilon-N membrane forhybridization with a nick-translated prostasin promoter probe (bases703-1469 of the prostasin gene sequence U33446). The probe detects apromoter fragment of 1,275 bp, which is cut by themethylationinsensitive enzyme Msp I to yield a 1,052-bp fragment for allDNA samples. The methylation-sensitive isoschizomer Hpa II yields the1,052-bp fragment in the CC-2555 (normal prostate epithelial cells), theLNCaP, and the MCF-7 samples, indicating the hypomethylated orunmethylated state of the prostasin promoter in these cells. For DU145,PC-3, and MDA-MB-453 DNA, both the 1,052-bp and the 1,275-bp fragmentsare generated in the methylation-sensitive digestion, suggestingincomplete methylation (one of two or more chromosomes) or clonalmethylation in a sub-population of cells. For the MDA-MB-231 andMDA-MB-435, however, the Hpa II digestion did not yield the 1,052 bp butrather gave the 1,275-bp fragment. This homogeneous methylation patternindicates that the Msp I/Hpa II site, at location -95 (relative to thetranscription initiation site) of the prostasin promoter, is methylated(hypermethylated) in these DNA samples. Signal intensity variation maybe attributed to aneuploidy.

[0055] An examination of the prostasin gene promoter region for DNAmethylation differences among human prostate and breast cancer celllines has been made.(see FIG. 6). We found that cells that expressprostasin, normal prostate epithelial, LNCaP, and MCF-7, areunmethylated in the prostasin promoter while MDA-MB-453 showedheterogeneous prostasin promoter methylation. For cells that do notexpress prostasin, DU-145 and PC-3 showed heterogeneous prostasinpromoter methylation while MDA-MB-231 and MDA-MB-435 showed homogeneoushypermethylation in the promoter region of the prostasin gene.

[0056] Two human prostate cancer cell lines that do not expressprostasin, the highly invasive DU-145 and PC-3, show heterogeneousmethylation in the promoter region of the prostasin gene. The resultsuggests that at least one of the two (or more) chromosome 16's of thesecell lines is methylated at the prostasin gene locus. The prostasin geneon the unmethylated chromosome may contain mutations that silenced theexpression. An alternative explanation for the heterogeneous methylationpattern is that the methylation occurs in clonal cell populations,however, the lack of detectable prostasin mRNA in our RT-PCR-Southernblot analysis in the DU-145 and PC-3 cells argues against thispossibility.

[0057] The significance of the finding on prostasin gene promoterhypermethylation in prostate or breast cancer is that the measurement ofprostasin down-regulation as a cancer marker may be achieved by using abinary assay (yes-or-no), instead of a gradually decreasing quantity inthe immunohistochemistry assay (which is quite arbitrary).

[0058] Re-expression of human prostasin protein in invasive humanprostate and breast cancer cells reduces invasiveness in vitro. At thispoint, reference should be made to FIG. 7 which shows the prostasinprotein expression and in vitro invasive properties of the DU-145 andthe PC-3 transfectants. DU-145 or PC-3 cells transfected with either avector DNA (labeled as “vector”) or a prostasin cDNA construct (labeledas “prostasin”) were analyzed by a western blot analysis using aprostasin-specific antibody (upper panel) or subjected to an in vitroMatrigel chemoinvasion assay (lower panel) as described in (Liu DF.Rabbani SA. Induction of urinary plasmiinogen activator by retina acidresults in increased invasiveness of human prostate cancer cells PC-3.Prostate. 27(5):269-76, 1995). The expressed human prostasin protein (a40-kDa band) was detected in the prostasin cDNA-transfected DU-145 orPC-3 cells, but not in the vector-transfected cells. In the Matrigelchemoinvasion assay, the vector-transfected cells are expressed as being100% invasive (solid bar), the open bar represents the relativeinvasiveness of the human prostasin cDNA-transfected cells. The datawere analyzed by a Student t-test using the Stat view software (AbacusConcepts, Inc., Berkeley, Calif.).

[0059] It can be seen that Polygonal DU-145 and PC-3 cells transfectedwith the human prostasin cDNA (designated DU-145/Pro, and PC-3/Pro,respectively) were confirmed to express the human prostasin protein, asshown in the western blot analysis of the cell lysate (FIG. 7, upperpanel). The vector-transfected cells, designated DU-145/Vector orPC-3/Vector, respectively, were used as negative control in the westernblot. A further examination of the DU-145/Pro and the PC-3/Pro cells byimmunocytochemistry confirmed that 100% of the cells expressed theprostasin protein (data not shown). In in vitro Matrigel chemoinvasionassays (FIG. 7, lower panel), the invasiveness of DU-145/Pro cells wasdetermined to be at 32% of that of DU-145/Vector cells (or, thereduction of invasiveness was at 68%). The invasiveness of PC-3/Procells was determined to be at 58% of that of PC-3/Vector cells (or, thereduction of invasiveness was at 42%). We performed in vitro cellproliferation assays on DU-145/Pro vs. DU-145/Vector cells, and onPC-3/Pro vs. PC-3/Vector cells, but did not observe any differencebetween the growth rates of the prostasin cDNA-transfected or thevector-transfected cells over an 8-day period (data not shown).

[0060] Forced re-expression of human prostasin in two invasive humanbreast cancer cell lines reduced invasiveness. A full-length humanprostasin cDNA under the control of an RSV promoter was transfected intothe invasive breast cancer MDA-MB-231 and MDAMB-435 cells. Referenceshould be made to FIG. 8 which shows prostasin protein expression and invitro invasive properties of the MDA-MB-231 and the MDA-MB-435stransfectants. MDA-MB-231 and MDA-MB-435s cells transfected with eithera vector DNA (labeled as “vector”) or a prostasin cDNA construct(labeled as “prostasin”) were analyzed by a western blot analysis usinga prostasin-specific antibody (upper panel) or subjected to an in vitroMatrigel chemoinvasion assay (lower panel) as referenced in (Liu DF.Rabbani SA. Induction of urinary plasminogen activator by retina acidresults in increased invasiveness of human prostate cancer cells PC-3.Prostate. 27(5):269-76, 1995). The expressed human prostasin protein (a40-kDa band) was detected in the prostasin cDNA-transfected MDA-MB-231and MDA-MB-435s cells, but not in the vector-transfected cells. In theMatrigel chemoinvasion assay, the vector-transfected cells are expressedas being 100% invasive (solid bar), the open bar represents the relativeinvasiveness of the human prostasin cDNA-transfected cells. The datawere analyzed by a Student t-test using the Stat view software (AbacusConcepts, Inc., Berkeley, Calif.).

[0061] Stable, polyclonal, episomal transfectants were obtained and theexpression of human prostasin protein was confirmed by western blotanalysis (FIG. 8, upper panel). In in vitro Matrigel chemoinvasionassays, the invasiveness of either cell lines expressing human prostasinwas reduced by 50% as compared to the vector-transfected controls (FIG.8, lower panel).

[0062] Taken together, the foregoing evidences linking prostasin levelreduction or protasin absence to the invasiveness of prostate and breastcancer cell lines, or linking prostasin expression to reducedinvasiveness. The evidence qualifies prostasin as an invasionsuppressor, which thus is a marker for diagnosis of invasiveness ofprostate and breast cancers, or as a therapeutic agent to treat invasiveprostate and breast cancers.

[0063] Pathological grading by the Gleason system is performed aftereither surgery or biopsy, both highly invasive procedures, while bloodtests such as that for the PSA prostate cancer marker can offer the hopeof accurate diagnosis and prognosis without the harm of an invasiveprocedure. In practice, however, single markers suffer from an intrinsiclimitation that the “positive” identifications are not always confirmedfor the “diagnosed” disease. Biopsy is still required for a truepositive identification of prostate cancer even in the case of theapplication of the PSA prostate cancer marker (Catalona WJ. Partin AW.Slawin KM. Brawer MK. Flanigan RC. Patel A. Richie JP. deKernion JB.Walsh PC. Scardino PT. Lange PH. Subong EN. Parson RE. Gasior GH.Loveland KG. Southwick PC. Use of the percentage of freeprostate-specific antigen to enhance differentiation of prostate cancerfrom benign prostatic disease: a prospective multicenter clinical trial[see comments]. JAMA. 279(19):1542-7, 1998). As stated above, it hasbeen demonstrated in principle that many markers used in a multivariateapproach may provide a highly accurate diagnosis (Greene GF. Kitadai Y.Pettaway CA. von Eschenbach AC. Bucana CD. Fidler IJ. Correlation ofmetastasis-related gene expression with metastatic potential in humanprostate carcinoma cells implanted in nude mice using an in situmessenger RNA hybridization technique. American Journal of Pathology.150(5):1571-82, 1997). From the standpoint of prostate cancer genetics,the multivariate approach is well supported by our currentunderstanding. A serine protease structurally and genetically related tothe PSA, the hK2 (human glandular kallikrein 2) has shown some promiseof joining the list of markers applicable for prostate cancer diagnosis(Saedi MS. Hill TM. Kuus-Reichel K. Kumar A. Payne J. Mikolajczyk SD.Wolfert RL. Rittenhouse HG. The precursor form of the human kallikrein2, a kallikrein homologous to prostate-specific antigen, is present inhuman sera and is increased in prostate cancer and benign prostatichyperplasia. Clinical Chemistry. 44(10):2115-9, 1998). Structurally andin prostate gland biology, prostasin shares many common characteristicswith both PSA and hK2, as being a secreted serine protease made in largeabundance in prostate epithelial cells (Yu JX. Chao L. Chao J. Prostasinis a novel human serine proteinase from seminal fluid. Purification,tissue distribution, and localization in prostate gland. Journal ofBiological Chemistry. 269(29):18843-8, 1994; Yu JX. Chao L. Chao J.Molecular cloning, tissue-specific expression, and cellular localizationof human prostasin mRNA. Journal of Biological Chemistry.270(22):13483-9, 1995). While highgrade prostate cancer cells produceless PSA protein than normal prostate cells or lowgrade prostate cancercells (Hakalahti L. Vihko P. Henttu P. Autio-Harmainen H. Soini Y. VihkoR. Evaluation of PAP and PSA gene expression in prostatic hyperplasiaand prostatic carcinoma using northern-blot analyses, in situhybridization and immunohistochemical stainings with monoclonal andbispecific antibodies. International Journal of Cancer. 55(4):590-7, 1;Sakai H. Yogi Y. Minami Y. Yushita Y. Kanetake H. Saito Y. Prostatespecific antigen and prostatic acid phosphatase immunoreactivity asprognostic; Sakai H. Yogi Y. Minami Y. Yushita Y. Kanetake H. Saito Y.Prostate specific antigen and prostatic acid phosphataseimmunoreactivity as prognostic), the serum PSA levels in prostate cancerpatients increase due to tissue damage caused by invasive cancer(Rittenhouse HG. Finlay JA. Mikolajczyk SD. Partin AW. Human Kallikrein2 (hK2) and prostate-specific antigen (PSA): two closely related, butdistinct, kallikreins in the prostate. [Review] [457 refs] CriticalReviews in Clinical Laboratory Sciences. 35(4):275-368, 1998). Bycomparison, we also believe prostasin is in the circulation of prostatecancer patients. As a result, a blood test for the circulating prostasinto indicate the presence and/or the stage of prostate cancer would behighly useful. (as illustrated in FIG. 1). In principal, the feasibilityof a blood test based on prostasin detection to indicate cancer has beendemonstrated by Berteau et al. (1999). These authors (Berteau P. LaribiEschwege P. Lebars I. Dumas F. Benoit G. Loric S. Prostasin mRNA todetect prostate cells in blood of cancer patients. Clinical and ChemicalLaboratory Medicine 37 (SS): S119, 1999), demonstrated a highlypromising potential of using prostasin as a marker to detect circulatingprostate epithelial cells, a sign of prostate tissue damage caused byinvasive prostate cancer leading to the dissemination of prostateepithelial cells into the circulation. It is expected that a blood testfor circulating prostasin to indicate the presence and/or the stage ofbreast cancer would be highly useful. (as illustrated in FIG. 2).

[0064] In summary of the invention, it has been taught herein that:protein prostasin as well as its MRA levels and its gene promoter DNAmethylation levels can be used to determine the invasiveness level ofhuman carcinomas; and, provide a method of treating invasive humancarcinomas by delivery thereto of a recombinant nucleic acid formed by ahuman prostasin nucleic acid incorporated into a selected gene deliveryvector. Those teachings are repeated for emphasis in the following:

[0065] 1. Immunohistochemistry studies of human prostate cancerspecimens revealed a down-regulation of prostasin in high-grade tumors.

[0066] 2. Using RT-PCR and western blot analyses, prostasin protein andmRNA expression were found in a non-invasive human breast cancer cellline, MCF-7, while invasive human breast cancer cell lines MDA-MB-231and MDA-MB435s were found not to express either the prostasin protein orthe mRNA. A non-invasive human breast cancer cell line, MDA-MB-453, wasshown to express prostasin mRNA but not prostasin protein: and,

[0067] 3. Examination of the prostasin gene promoter in the humanprostate and breast cancer cell lines by Southern blot analysis revealedheterogeneous methylation of the promoter in DU-145, PC-3 and MDA-MB-453cells, and homogeneous methylation of the promoter in MDA-MB-231 andMDA-MB-435s cells. The prostasin gene promoter in normal human prostateepithelial cells, the LNCaP and the MCF-7 cells was shown to beunmethylated. Transfection of DU-145 and PC-3 cells with a full-lengthhuman prostasin cDNA restored prostasin expression and reduced the invitro invasiveness by 68% and 42%, respectively. Transfection ofMDA-MB-231 and MDA-MB-435s cells with a full-length human prostasin cDNArestored prostasin expression and reduced the in vitro invasiveness by50% for either cell line.

[0068] The preferred methods of separating sampled human carcinomatissue from neighboring normal tissues is by laser capturemicro-dissection.

[0069] While the invention has been described, disclosed, illustratedand shown in various terms of certain embodiments or modifications whichit has presumed in practice, the scope of the invention is not intendedto be, nor should it be deemed to be, limited thereby and such othermodifications or embodiments as may be suggested by the teachings hereinare particularly reserved especially as they fall within the breadth andscope of the claims here appended.

We claim:
 1. A method of determining invasiveness levels of humancarcinomas using prostasin protein levels, comprising the steps of:sampling a human carcinoma tissue; determining prostasin protein levelsin the human carcinoma tissue; and determining invasiveness of the humancarcinoma tissue based on the prostasin protein levels.
 2. The method ofdetermining invasiveness levels of claim 1, wherein the step ofdetermining prostasin protein levels includes: applying an immunologicalreagent-antibody to the sampled human carcinoma tissue, wherein thereagent-antibody becomes bound to prostasin protein in the humancarcinoma tissue. 3 . The method of determining invasiveness levels ofclaim 1, wherein the sampled human carcinoma tissue includes: prostatecancer tissue.
 4. The method of determining invasiveness levels of claim1, wherein the sampled human carcinoma tissue includes: breast cancertissue.
 5. A method of determining invasiveness levels of humancarcinomas using prostasin mRNA levels, comprising the steps of:sampling a human carcinoma tissue; determining prostasin mRNA levels inthe human carcinoma tissue; and determining invasiveness of the humancarcinoma tissue based on the prostasin mRNA levels.
 6. The method ofdetermining invasiveness levels of claim 5, wherein the step ofdetermining prostasin mRNA levels includes: applying prostasin-specificoligonucleotide primers in an RT-PCR to determine the prostasin mRNAlevels in the sampled human carcinoma tissue.
 7. The method ofdetermining invasiveness levels of claim 5, wherein the sampled humancarcinoma tissue includes: prostate cancer tissue.
 8. The method ofdetermining invasiveness levels of claim 5, wherein the sampled humancarcinoma tissue includes: breast cancer tissue.
 9. The method ofdetermining invasiveness levels of claim 5, wherein the sampling of thehuman carcinoma tissue includes the step of: separating the humancarcinoma tissue from neighboring normal tissues.
 10. The method ofdetermining invasiveness levels of claim 9, wherein the separating stepincludes: a laser capture micro-dissection.
 11. The method ofdetermining invasiveness levels of claim 5, wherein the step ofdetermining prostasin mRNA levels includes: applying prostasin-specificanti-sense RNA probes in an in situ hybridization to determine theprostasin mRNA levels in the sampled human carcinoma tissue.
 12. Amethod of determining invasiveness levels of human carcinomas usingprostasin gene promoter DNA methylation levels, comprising the steps of:sampling a human carcinoma tissue; determining prostasin gene promoterDNA methylation levels in the human carcinoma tissue; and determininginvasiveness of the human carcinoma tissue based on the prostasin genepromoter DNA methylation levels.
 13. The method of determininginvasiveness levels of claim 12, wherein the step of determining genepromoter DNA methylation levels includes: applyingprostasin-promoter-specific oligonucleotide primers in a PCR todetermine the prostasin gene promoter DNA methylation levels in thesampled human carcinoma tissue.
 14. The method of determininginvasiveness levels of claim 13, wherein the sampling of the humancarcinoma tissue includes the step of: separating the human carcinomatissue from neighboring normal tissues.
 15. The method of determininginvasiveness levels of claim 14, wherein the separating step includes: alaser capture micro-dissection.
 16. The method of determininginvasiveness levels of claim 12, wherein the step of determining genepromoter DNA methylation levels includes: applyingprostasin-promoter-specific DNA probes in a Southern blot hybridizationanalysis to determine the prostasin gene promoter DNA methylation levelsin the sampled human carcinoma tissue.
 17. The method of determininginvasiveness levels of claim 16, wherein the sampling of the humancarcinoma tissue includes the step of: separating the human carcinomatissue from neighboring normal tissues.
 18. The method of determininginvasiveness levels of claim 17, wherein the separating step includes: alaser capture micro-dissection.
 19. The method of determininginvasiveness levels of claim 12, wherein the sampled human carcinomatissue includes: prostate cancer tissue.
 20. The method of determininginvasiveness levels of claim 12, wherein the sampled human carcinomatissue includes: breast cancer tissue.
 21. A method of treating invasivehuman carcinomas, comprising the steps of: incorporating human prostasinnucleic acid into a selected gene delivery vector nucleic acid to form arecombinant nucleic acid; delivering the recombinant nucleic acid into ahuman carcinoma; and reducing invasiveness of the human carcinoma. 22.The method of treating invasive human carcinomas of claim 21, whereinthe nucleic acid includes: a gene.
 23. The method of treating invasivehuman carcinomas of claim 21, wherein the nucleic acid includes: a cDNA.24. The method of treating invasive human carcinomas of claim 21,wherein the human carcinoma includes: prostate cancer.
 25. The method oftreating invasive human carcinomas of claim 21, wherein the humancarcinoma includes: breast cancer.